ELPs, as explained more fully in the Detailed Description a the Invention hereof (Section 5) are oligomeric repeats of the pentapeptide Val-Pro-Gly-X-Gly (Sequence ID No. 1), here the guest residue X is any amino acid. ELPs undergo a reversible inverse temperature transition. They are highly soluble in water below the inverse transition temperature (Tt), but undergo a sharp (2-3° C. range) phase transition when the temperature is raised above their Tt, leading to desolvation and aggregation of the polypeptide.1,2,3 In previous work, McPherson et al. have exploited the inverse transition to purify recombinant poly(GVGVP) polypeptide. Previous studies have also shown that protein conjugates of poly(N-isopropylacrylamide), a synthetic polymer that undergoes a similar thermally-reversible phase transition, also retain the transition behavior of the free polymer.5,6,7 
Recombinant DNA techniques have facilitated the expression of proteins for diverse applications in medicine and biotechnology. However, the purification of recombinant proteins is often complicated and problematic. In the last decade, a number of protein expression systems have been developed to simplify protein purification.8,9 Such systems often operate by expressing a recombinant protein fused with a carrier protein or peptide. A number of fusion protein systems using different carrier proteins are now commercially available, particularly for E. coli expression. Examples include maltose binding protein,10 glutathione S-transferase,11 biotin carboxyl carrier protein,12,13 thioredoxin,13,14 and cellulose binding domain.15 Similarly, vectors that allow fusion of the target protein to short peptide tags such as oligohistidine,16 S-peptide,17 and the FLAG peptide18 are also available.
Fusion protein expression simplifies the separation of recombinant protein from cell extracts by one-step purification by affinity chromatography using an immobilized, moderate-affinity ligand specific to the carrier protein.19 Although useful for laboratory scale purification, the scale-up of affinity chromatography can represent a major cost of the final protein product at the preparative scale.
Additionally, chromatography represents a major bottleneck in high throughput purification of proteins. The full implications of the human genome project will not be realized until all the proteins encoded in the genome can be expressed and studied in detail. Current chromatographic technologies cannot be easily multiplexed to efficiently purify the wide diversity of proteins encoded in the human genome. These limitations of current bioseparation techniques, therefore, provide a compelling rationale for the development of nonchromatographic methods for the purification of soluble, recombinant proteins. Likewise, nonchromatographic purification methods would also be attractive as technically simple, reliable, and broadly applicable methods for bench top, milligram-scale purification of single proteins.
More economical and technically simple methods for purification of soluble proteins, which do not involve scale-up of chromatographic procedures, are therefore desirable.
The inventor has surprisingly discovered that incorporation of ELPs in fusion proteins, enables non-chromatographic, thermally-stimulated phase separation of recombinant proteins. These FPs undergo a phase transition similar to that of free ELPs. This surprising discovery is useful in the purification of fusion proteins incorporating one or more ELP tags.
Elastin-like polypeptides (ELPs) are thermally responsive polypeptides that undergo reversible aggregation above a critical temperature. An ELP is a polypeptide with the repeating pentapeptide sequence Val-Pro-Gly-Xaa-Gly (where the guest residue Xaa can be any amino acid, except Pro, in any fraction). Below the inverse transition temperature (Tt), ELPs are structurally disordered and soluble in aqueous solutions, but when the temperature is raised above their Tt, they undergo a sharp (2-3° C. range) disorder-to-order transition, leading to desolvation and aggregation of the polypeptide. These ELP aggregates can have sufficient mass that they can be removed from solution by centrifugation, but because the inverse transition is reversible, they can be completely resolubilized in buffer when the temperature is returned below the Tt of the polypeptide.
By fusing a thermally responsive ELP to a target protein of interest, environmentally sensitive solubility can be imparted to the target protein. In the practice of the present invention, target proteins are expressed as soluble fusion proteins with N- or C-terminal ELP sequences in organisms such as E. coli, wherein the fusion proteins exhibit a soluble-insoluble phase transition. This inverse phase transition is exploited in the process of the invention to purify the fusion proteins from other soluble proteins produced by the organism, using a new nonchromatographic separation method for recombinant proteins, which the present inventor has termed “inverse transition cycling” (ITC).
The fundamental principle of ITC is remarkably simple. It involves rendering the ELP fusion protein insoluble in aqueous solution by triggering its inverse transition. This can be accomplished either by increasing the temperature above the Tt, or alternatively by depressing the Tt below the solution temperature by the addition of NaCl to the solution. This results in aggregation of the ELP fusion protein, allowing it to be collected by centrifugation. The aggregated fusion protein can then be resolubilized in fresh buffer at a temperature below the Tt, thereby reversing the inverse transition, to yield soluble, functionally active, and purified fusion protein.
Free target protein then can be obtained, for example, by protease digestion at an engineered recognition site, located between the target protein and the ELP tag, followed by a final round of ITC to remove the cleaved ELP.
ITC has major advantages over other methods currently used for purification of recombinant proteins. It is technically simple, inexpensive, easily scaled up, and gentle, triggered by only modest alterations in temperature and/or ionic strength. The ITC technology is useful in the modulation of the physico-chemical properties of recombinant proteins and provides diverse applications in bioseparation, immunoassays, biocatalysis, and drug delivery.
The ITC methods of the invention exhibit significant advantages over currently used affinity purification methods in purifying recombinant fusion proteins. First, by circumventing chromatography, the expense associated with chromatographic resins and equipment is eliminated. Second, the separation and recovery conditions are gentle, requiring only a modest change in temperature or ionic strength. Third, the method is fast and technically simple, with only a few short centrifugation or filtration steps followed by resolubilization of the purified protein in a low ionic strength buffer. Finally, the equipment required, a temperature-controlled water bath and a centrifuge capable of operating at ambient temperature, are widely available. Additionally, ITC purification is independent of a specific expression vector or host and is exceptionally advantageous for use with eukaryotic expression systems, which readily over-express heterologous proteins in a soluble state.20 
The ITC methodology of the invention also addresses a compelling need in the art for high-throughput purification techniques. The ITC purification technique of the invention is scalable in character, and can be appropriately scaled and multiplexed for concurrent, parallel laboratory purifications from numerous cell cultures.
Simultaneous purification of proteins from multiple cultures using the ITC methodology of the invention enables expedited structure-function studies of proteins as well as screening of proteins in pharmaceutical studies.